Project 3 (P3). Noncanonical activities of a tRNA synthetase in metabolism and atherosclerosis Paul L. Fox, Ph.D., Project Leader Project Summary/Abstract The long-term goal of Project 3 is to understand the noncanonical function of an extraordinary tRNA synthetase, the Glu-Pro tRNA synthetase or EPRS, in diet-induced obesity and consequent cardiovascular disease, particularly atherosclerosis. The recent explosive epidemic of obesity, insulin resistance, and cardiovascular disease has begun an unprecedented decline in the health-span of adults in the U.S., and likewise threatens an equally unprecedented economic burden. In 1942, James Neel suggested a possible rationale for the genetic selection of genes causing these pathologies. He posited that metabolic pathways were naturally selected to efficiently store fat and carbohydrate during periods of food scarcity; however, the same genes and pathways are detrimental during periods of plentiful, calorie-rich food supply as in the Western world today. A kinase cascade involving the mammalian target of rapamycin (mTORC1) and ribosomal protein S6 kinase-1 (S6K1) is implicated as a key metabolic pathway regulating food utilization, and is conserved from Drosophila to humans. Despite intense study, the key downstream effector(s) of mTORC1-S6K1, and consequent cell mechanisms that regulate metabolism remains unknown. During the previous Project period, we made fundamental in vitro and in vivo discoveries that revealed phosphorylated EPRS as a key downstream effector of mTORC1-S6K1, regulating post-transcriptional, inflammation-related pathways in macrophages and metabolic pathways in adipocytes. Inflammatory macrophages permeate the more abundant adipocytes in adipose tissue of obese subjects, and EPRS phosphorylation in both cell types by S6K1 might functionally couple these cells, and contribute importantly to obesity-associated cardiovascular disease. To test the role of phospho-EPRS in these processes we have generated genetically-modified EPRS phospho-deficient and phospho-mimetic knock-in mice. Preliminary studies show that phospho-deficient EPRS mice phenocopy S6K1-null mice, e.g., small size and low fat mass, and will permit rigorous investigation of the role of EPRS in mTORC1-S6K1-driven mechanisms in vivo. We propose to test the following hypothesis: Phospho-EPRS is a critical effector of the mTORC1-S6K1 signaling pathway in both adipocytes and macrophages, and contributes importantly to diet- induced obesity and atherosclerosis. Our discovery that EPRS is a key mTORC1-S6K1 effector activated by agonists of both inflammation and metabolism provides a molecular link between these processes. We anticipate our studies will reveal new mechanisms underlying obesity and atherosclerosis, and can provide novel therapeutic targets for treatment of these related disorders.